Materials and methods for the enhancement of effective root nodulation in legumes

ABSTRACT

The subject invention relates to compounds and compositions which induce transcription of the nolA gene in nitrogen-fixed bacteria, such as  Bradyrhizobium japonicum.  Novel bacterial strains which are insensitive to NolA, soil inoculants comprising NolA insensitive bacteria and/or nolA inducers, and methods of increasing nitrogen fixation in legumes are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. application Ser.No.09/909,735, filed Jul. 20, 2001, which claims priority to U.S.Provisional Application No. 60/219,509, filed Jul. 20, 2000. Each ofthese applications are hereby incorporated by reference in its entirety,including the disclosure, figures, tables, or drawings.

[0002] The subject invention was made with government support under aresearch project supported by The National Science Foundation Grant No.IBN-9728281. The government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Leguminous plants, such as soybeans, are able to fix nitrogenfrom the atmosphere due to a symbiotic relationship between the plantsand bacteria which dwell in nodules formed in the roots of the plants.Specifically, soil bacteria that are members of the family Rhizobiaceae,are capable of infecting plants and inducing highly differentiated rootnodule structures within which atmospheric nitrogen is reduced toammonia by the bacteria. The host plant utilizes the ammonia as a sourceof nitrogen. The symbiotic root nodule bacteria are classified inseveral separate genera, including Rhizobium, Bradyrhizobium,Sinorhizobium, and Azorhizobium.

[0004] Legume nodulation by rhizobia exhibits some species specificity.Bradyrhizobium species include the commercially important soybeannodulating strains B. japonicum (i.e., strains USDA110 and 123),promiscuous rhizobia of the cowpea group, and B. parasponia (formerlyParasponia rhizobium) which nodulates the non-legume Parasponia, as wellas a number of tropical legumes, including cowpea and siratro. The mostimportant agricultural host of B. japonicum is soybean (Glycine max),but this bacterium will nodulate a few other legumes (e.g., cowpea andsiratro). Fast growing rhizobia include, among others, Rhizobium etli,Sinorhizobium meliloti (formerly Rhizobium meliloti), and Rhizobiumleguminosarum biovar trifolii, which nodulate bean, alfalfa, and clover,respectively. These Rhizobium species generally display a narrow hostrange. However, Rhizobium sp. NGR234 has the ability to nodulate over100 genera of legumes. Sinorhizobiium fredii (formerly Rhizobiumfredii), is phylogenetically distinct from B. japonicum, but has theability to nodulate Glycine soja (a wild soybean species), G. max cv.Peking, and a few other soybean cultivars.

[0005] There are currently about 70,000,000 acres of soybean grown inthe United States. An inoculant industry exists to sell B. japonicum tofarmers for incorporation into the soil during soybean planting. The useof these inoculants is intended to enhance the efficiency of nitrogenfixation. Unfortunately, for most of the United States, inoculation hasbeen shown to be ineffective. Therefore, the inoculant industry remainsrelatively small (approximately $20-30 million per year). Indeed, atpresent, inoculation is only recommended for newly planted fields (i.e.,those not planted with soybeans previously) and fields that have beenout of production for over three years.

[0006] The primary reason for the inefficiency of soil inoculation isthe presence of competing extant B. japonicum in soil. When a field hasbeen producing soybean for more than one season, there is a build up ofthe B. japonicum populations in soil. These bacteria are highlycompetitive since they have adapted to their soil environment. Hence,when the inoculant is added, the indigenous soil B. japonicum strainscompete and win the battle to nodulate the plant. The result is that, inmany cases, less than 1% of the nodules formed on the planted soybeanare due to the inoculant added. Therefore, even if a high-yielding B.japonicum strain is used as the inoculant, the farmer does not see theyield increase due to the fact that the inoculant has not found its wayinto the plant.

[0007] In the major soybean growing areas of the Midwest, the mostcompetitive population of B. japonicum is that of serogroup 123. Ifimprovement in the nitrogen fixing capacity of thesoybean-Bradyrhizobium symbiosis through application of superior strainsis to be realized, then the difficult problem of competition fromindigenous populations (such as serogroup 123) will have to be solved.

[0008] Significant efforts have been made to understand and alter thecompetitiveness of indigenous Bradyrhizobia. For example, attempts toalter soybean nodule occupancy ratios of indigenous versus appliedBradyrhizobia have been reported. However, such alterations were onlyachieved by using ultra-high, economically infeasible rates of theapplied strain. In a seven year study, Dunigan et al. [Agron. J. 76:463-466 (1984)] demonstrated that the inoculant strain USDA 110eventually formed the majority of nodules after high rates ofapplication in the first 2 years (serogroup 123 was not among theindigenous population). However, the tenacious competitive ability ofserogroup 123 appears not to be related to numbers per se and whennormal rates of inoculant are applied the indigenous serogroup 123population can still form up to 95% of the nodules on soybean.

[0009] The formation of nodules on leguminous plants involves a complexexchange and recognition of diffusible signals between the plant and thebacterial symbiont. A key plant signal are the flavonoids which triggerthe induction of the bacterial nodulation genes (Day et al [2000] In:Prokaryotic Nitrogen Fixation: A Model System for the Analysis of aBiological Process, ed. Triplett, E., Horizon Scientific Press, Norfolk,England, pp 385-414).

[0010] Nodulation genes of Bradyrhizobium and Rhizobium strains affectthe early stages of nodule formation including host-bacteriumrecognition, infection and nodule development. Wild type strains ofBradyrhizobium species display some variation in these early nodulationsteps which is reflected in differences in relative rates of initiationof nodulation and ultimately in differences in competitiveness betweenstrains for nodule occupancy. For example, B. japonicum USDA 123 isbelieved to be more competitive for nodulation than B. japonicum USDA110. Strains which initiate infection and nodules earlier will occupy agreater portion of the nodules on a given plant. Improving thecompetitiveness of a specific Bradyrhizobium is an important part of thedevelopment of improved inoculants for legumes. A more effectiveBradyrhizobium strain must be able to out-compete the indigenousrhizobia population for nodule occupancy in order for their improvedqualities to impact on the inoculated legume. Therefore, there is asignificant need for an inoculating composition and/or an inoculatingmethod which would improve competitiveness of a selected inoculantstrain.

[0011] In the Bradyrhizobium japonicum-soybean symbiosis, several keyregulatory components have been identified in the regulation ofbacterial nodulation genes. Two of these, i.e., a LysR regulator, NodD₁and a two component regulatory system, NodWV are known to positivelyactivate the B. japonicum nodulation genes in response to the plantproduced isoflavonoids, genistein and daidzein. A third regulatorycomponent (i.e., NolA) is a MerR type regulator (Sadowsky et al. [1991]Proc. Natl. Acad Sci. USA 88:637-641) that possesses the unique capacityto exist in three functionally distinct forms (i.e., NolA₁, NolA₂ andNolA₃) (Loh et al. [1999] J. Bacteriol. 181:1544-1554). Thesepolypeptides are derived from alternative translation of three in-frameinitiation codons.

[0012] Induction of the B. japonicum nola gene leads to the subsequentrepression of the nodulation genes in this bacterium. The products ofthe nodulation genes are required for soybean nodulation. Thus, theseplant compounds, by inducing nolA expression, lead eventually to aninhibition of nodulation.

[0013] NolA, is required for the expression of both NolA₂ and NolA₃. Twotranscriptional (P1 and P2) start sites have been identified (Loh et al.[1999] J. Bacteriol. 181:1544-1554). Transcription from P1 results inthe formation of an mRNA encoding NolA₁. NolA₁ then regulatestranscription from P2, resulting in the expression of both NolA₂ andNolA₃.

[0014] Although NolA is involved in the negative control of thenodulation genes (Dockendorff, T. C., J. Sanjuan, P. Grob, and G. Stacey[1994] Mol. Plant-Microbe Interact. 7:596-602), current informationsuggests that NolA does not act directly to repress nod gene expression.This view is supported by the observation that while expression of NolAfrom a multicopy plasmid resulted in a reduction of nod gene expression,interposon mutations to the nolA gene did not lead to elevated levels ofnod gene expression (Garcia, M. L., J. Dunlap, J. Loh, and G. Stacey[1996] Mol. Plant-Microbe interact 9:625-635). In fact, NolA appears topositively regulate the expression of NodD₂, the latter of which hasbeen shown to be a repressor of the nod genes in Rhizobium spp. NGR234,Bradyrhizobium spp. (Arach is) NC92 and Bradyrhizobium japonicum(Garcia, M. L., J. Dunlap, J. Loh, and G. Stacey [1996] Mol.Plant-Microbe Interact 9:625-635; Gillette, W. K. and G. H. Elkan [1996]J. Bacteriol. 178:2757-2766; and Fellay, R., M. Hanin, G. Montorzi, J.Frey, C. Freiberg, W. Golinowski et al. [1998] Mol. Microbiol.27:1039-1050. Therefore, NolA affects repression indirectly, through thecontrol of nodD₂ expression.

[0015] Cell-cell signaling plays a large role in the ability of bacteriato respond and adapt to a particular environment. Regulatory systemsthat control gene expression in response to population density (i.e.,quorum sensing) govern such bacterial phenotypes as bioluminescence,antibiotic production, plasmid conjugal transfer and the synthesis ofvirulence factors in both plant and animal pathogens (Hardman, A. M. etal. [1998] Antonie van Leeuwenhoek 74:199-210). Quorum sensing involvesthe recognition of self-produced signal compounds, which function toregulate the expression of genes when threshold levels of these signalshave accumulated in cultures of a sufficiently high population density.In Gram-negative bacteria, the best studied of these signals are N-Acylhomoserine-lactones (AHL) (Fuqua, W. C. et al. [1994] J. Bacteriol.176:269-275). In Gram-positive bacteria, an equivalent role is played byvarious posttranslationally-modified peptides (Kleerebezem, M. et al.[1997] Mol. Microbiol. 24:895-904). Several AHL compounds have beenidentified from rhizobia, including Rhizobium leguminosarum biovarsviciae, trifolii and phaseoli, Rhizobium etli, and Rhizobium meliloti(Thome and Williams [1999] J. Bacteriol. 181:981-990; Cha et al. [1998]Mol. Plant Microbe Int. 11:1119-1129; Gray et al. [1996] J. Bacterial.178:372-376; Rosemeyer et al. [1998] J. Bacteriol. 180:815-821;VanBrussel et al. [1985] J. Bacteriol. 162:1079-1082; and Wijffelman etal. [1983] Mol. Gen. Genet. 192:171-176). In a few cases, theseautoinducers have been implicated in the nodulation process. Forexample, the small AHL molecule produced by R. leguminosarum bv. viciaeis required for the expression of the rhiABC operon, which is importantfor rhizosphere growth and nodulation efficiency (Cubo et al. [1992] J.Bacteriol. 174:4026-4035). In R. etli, mutations that disrupt AHLsynthesis resulted in decreased nodule numbers on host plants (Rosemeyeret al. [1998] J. Bacteriol. 180:815-821). Therefore, AHL-mediated quorumsensing may play an important role in the symbiotic process. To date, noquorum-sensing compound has been identified from the soybean symbiontBradyrhizobium japonicum.

[0016] The current invention addresses the inefficiency of soilinoculation due to the presence of competing indigenous B. japonicum insoil and provides novel compounds and compositions which increase theefficiency of nodulation in target plants. Specifically, fieldinoculants comprising high-yielding NolA insensitive B. japonicum andnolA inducers address the long standing obstacle of inefficientnodulation due to indigenous B. japonicum strains.

BRIEF SUMMARY OF THE INVENTION

[0017] The subject invention provides materials and methods to improvenitrogen fixation in leguminous plants. In a preferred embodiment of thesubject invention, the improvement in nitrogen fixation is achieved byproviding an inoculant of nitrogen-fixing bacteria which, when appliedaccording to the subject invention, have a competitive advantage overindigenous strains.

[0018] In a specific embodiment, the subject invention providescompounds and compositions which induce transcription of the nolA genein nitrogen-fixing bacteria, such as Bradyrhizobium japonicum. Byapplying these NolA inducers to the situs of indigenous B. japonicum itis possible to induce transcription of the nolA gene in indigenousbacteria, thereby reducing the ability of these bacteria to initiatenodulation.

[0019] A further aspect of the subject invention is the identificationof novel bacteria which are insensitive to NolA. In a preferredembodiment of the subject invention, these NolA insensitive microbes canbe applied to legumes in conjunction with the NolA inducers of thesubject invention. The NolA inducers inhibit the indigenous bacteria butdo not adversely affect the nodulation capabilities of the NolAinsensitive (NolA^(INS)) inoculant bacteria. This gives the inoculantbacteria a competitive advantage compared to the indigenous bacteria.

[0020] A further aspect of the subject invention relates to nucleicacids, expression cassettes, and vectors which encode the NolA inducercompounds of the subject invention. These genetic materials can be usedto efficiently produce the inducer compounds. The inducer compounds canbe produced in recombinant hosts including plants. Thus, one aspect ofthe subject invention concerns plants having polynucleotides whichencode compounds which induce transcription of the nolA gene innitrogen-fixing bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1—HPLC reverse phase chromatography (C18) of soybeanseedlings extract (SSGE) fractions. Peaks collected as IND-1 and IND-2were active on B. japonicum nolA-lacZ fusions.

[0022]FIG. 2A—Structure of IND-1. FIG. 2B—Effect of phthalic acidbis-(2-ethyl-hexyl) ester on nolA expression. B. japonicum cellsharboring a nolA-lacZ fusion were treated with increasing concentrationsof the phthalate derivate, and the level of nolA expression determined.

[0023]FIG. 3—Effect of phosphatidyl inositol extract on nola expression.Phosphatidyl inositol samples were either treated or untreated with 100μg/ml chitinase (Sigma Chemical Co.).

[0024]FIG. 4—Analysis of soybean phosphatidyl-inositol extracts. FIG.4A—Reverse phase comparison of extracts that had been untreated ortreated with chitinase (100 μg/ml). FIG. 4B—Effect of chitinase on theability of peak 9 to induce nolA expression.

[0025]FIG. 5—FIG. 5A depicts population density dependent expression ofnolA_(1,2,3)-lacZ and nodD₂-lacZ. B. japonicum cultures harboring eithernolA_(1,2,3)-lacZ nodD₂-lacZ or npt-lacZ were grown to variouspopulation densities and the β-galactosidase activity of these fusionsdetermined. Percent maximum activity is [β-galactosidaseactivity/maximal β-galactosidase activity of fusion]×100%. FIG.5B—Inducer of nolA expression is population density dependent.Conditioned medium was obtained from B. japonicum cultures grown tovarious population densities and used to induce a B. japonicum strainharboring a nolA_(1,2,3)-lacZ fusion. Standard deviation was less than10%.

[0026]FIG. 6—The inducibility of nod gene expression as a function ofinitial population density.

[0027]FIG. 7—Comparison of nodY-lacZ expression in a USDA110 and BjB3(nolA mutant). B. japonicum cultures were grown to various populationdensities and the ability of 0.05 μM genistein to induce nodY expressiondetermined. The fold induction is presented. The uninduced levels ofnodY-lacZ expression in USDA110 and BjB3 were 4±1 and 3±1, respectively.Standard deviation was less than 10%.

[0028]FIG. 8—Effect of IND-1 on genistein induction of a nodY-lacZexpression in B. japonicum. B. japonicum cells harboring a nodY-lacZfusion were incubated with increasing amounts and the ability of thiscompound to affect nod gene expression determined.

[0029]FIG. 9—Effect of IND-1 on nolA_(1,2,3), nolA₁, nolA₂ and nolA₃expression.

[0030]FIG. 10—Effect of quorum factor (i.e., conditioned medium) andIND-1 on the ability of B. japonicum strain USDA110 to nodulate soybean.B. japonicum cells were untreated (left) or incubated in conditionedmedium or IND-1 for 1 h, and then inoculated onto soybean plant (107cells per root). The number of nodules (±standard error) was determined21 days post-inoculation, both above the mark (i.e., upper zone), orbelow the mark (new tissue) at the time of inoculation (n=number ofplants per treatment).

[0031]FIG. 11A-C—The expression of nodD₂-lacZ and nolA-lacZ fusions as afunction of B. japonicum culture density was examined (FIG. 11A). NolA₁expression is cell-density dependent and required for NodD₂ expression(FIG. 11B). The ability of the conditioned medium to induce the nolAfusions was population density dependent with little or no induction ofthe fusions observed using conditioned medium derived from cultures ofA₆₀₀<0.2 (FIG. 11C).

[0032]FIG. 12—HPLC isolation of Cell Density Factor (Quorum Factor) fromB. japonicum conditioned medium (concentrated approximately 10-fold).Quorum factor containing material was applied to a C18 column(Phenomenex, Inc., Torrance, Calif.) and eluted with a methanol gradient(0-100%) at a flow rate of 1 mL per minute. Cell density factor wasdemonstrated to be a potent inducer of nolA expression.

[0033]FIG. 13—FIG. 13 provides a graphical depiction of the invention.

[0034] FIGS. 14A-B illustrate the effect of FeCl₃ on nolA-lacZexpression. B. japonicum cells harboring a nolA-lacZ fusion were treatedwith increasing concentrations of FeCl₃ for five hours and the level ofnolA expression was determined (FIG. 14A). FIG. 14B demonstrates anincrease in the expression of a nodY-lacZ fusion protein when cells aregrown in the presence of iron. B. japonicum cells containing a nodY-lazZfusion were induced for five hours with 0.025 μM genistein in thepresence or absence of 500 μM FeCl₃.

[0035]FIG. 15 shows the effect of bis-(2-ethyl-hexyl) ester phthlate(BEHP) on nodule occupancy by the NwsB mutant. Different ratios of B.japonicum USDA110 and B. japonicum NwsB mutant were innoculated onsoybean plants grown in growth pouches. At the time of innoculation, theroot tip (RT) mark was noted on the outside of the pouches. Nodules wereextracted 21 days post innoculation and the extracts were plated on RDYplates. Single colonies were picked and tested for streptomycinresistance (a marker for the NwsB mutant). (A) is above RT at time ofinnoculation; (B) below RT at time of innoculation.

[0036]FIG. 16 shows a mutant selection scheme for the isolation of B.japonicum mutants that nodulate in the presence of inhibitoryconcentrations of BEHP.

[0037] FIGS. 17-18 illustrates the expression of CDF or quorumfactor-like molecules in a variety of other bacteria.

DETAILED DISCLOSURE OF THE INVENTION

[0038] The subject invention provides materials and methods forpromoting the growth of leguminous plants by enhancing the efficiency ofroot nodulation by nitrogen-fixing bacteria. This enhancement ofnodulation efficiency is achieved by providing high-performing inoculantbacteria with a competitive advantage over indigenous bacteria.

[0039] Although indigenous bacteria are typically excellent competitorsfor forming root nodules, they are typically less efficient atnitrogen-fixation than inoculant bacteria. Therefore, in order for theinoculant bacteria to be capable of exerting their excellentnitrogen-fixing effects, they must first be able to out-compete theindigenous bacteria in order to form root nodules. Advantageously, thesubject invention provides materials and methods which enable theinoculant bacteria to establish root nodules, even in the presence ofindigenous bacteria.

[0040] In one aspect, the present invention provides isolated novelcompounds which induce transcription of the nolA gene. These compoundsare, collectively, referred to as nolA inducers. In soybean extracts,HPLC analysis of the compounds revealed at least two active compounds,referred to herein as IND-1 and IND-2. IND-1 has been identified asphthalic acid bis-(2-ethyl-hexyl) ester and is able to induce nolA.IND-1 has been identified as a contaminant of solvents used in theextraction process; however, phthalic acid bis-(2-ethyl-hexyl) ester isa potent inducer of nolA. IND-2 is a plant-produced NolA inducer thatcan be isolated according to the methods disclosed herein.

[0041] In addition to the plant-derived NolA inducers, the instantinvention also provides isolated novel compounds produced by B.japonicum which induce nolA expression. These novel compounds may alsobe referred to as bacterial nolA inducers. The bacterial nolA inducerappears to be produced in a density-dependent manner in batch cultureand may be referred to as a “quorum sensing” molecule or cell densityfactor (CDF). Quorum sensing molecules regulate the expression of genes,such as nolA, in response to bacterial population density. The bacterialnolA inducer is insensitive to heat treatment and appears to have amolecular weight of less than 3,000 Da.

[0042] Compositions comprising one or more nolA inducers and a carrierare also taught according to the subject invention. NolA inducersinclude chemical compounds, plant-derived NolA inducers, andbacterial-derived NolA inducers. By way of example, compositions havinga NolA inducer include commercially available soybean phosphatidylinositol extracts, conditioned medium obtained from cultured B.japonicum, commercially available soybean extracts, or compositionshaving IND-1 (or isomers, analogs, or homologs thereof), IND-2, or CDF.The compositions may, optionally, include one or more nolA^(INS)mutants.

[0043] Carriers useful in formulation of the compositions of theinvention are well known to those skilled in the art and include thosedescribed in detail in a number of sources which are well known andreadily available to those skilled in the art. Also contemplated ascarriers are agricultural materials such as soil additives. Non-limitingexamples of such additives include peat, soil conditioners, chemicalfertilizers, and organic fertilizers (such as chicken or cow manure).

[0044] The present invention also provides bacterial cells which areinsensitive to the effects of the nolA inducers. These bacterial cellsare referred to as NolA^(INS) mutants. An exemplary nolA^(INS) mutanthas been isolated and will be deposited with the American Type TissueCulture [10801 University Blvd., Manassas, Va. 20110-2209].

[0045] Other NolA^(INS) mutants include bacterial cells in which thegene or genes encoding the nolA inducer has been inactivated.Inactivation of the gene or genes encoding nolA inducers may beaccomplished by deletion of all, or a portion, of the gene or genesencoding the nolA inducer, insertion of nucleic acid sequences withingene or genes encoding the nolA inducer or inactivation oftranscriptional control sequences operably linked to nolA inducers.Alternatively, the nolA inducer gene may be inactivated by mutation ordeletion of ribosome binding sites. Mutation or deletion of translationinitiation sites may also be used to inactivate the nolA gene. Methodsof site directed mutagenesis in Gram negative bacteria, such asRhizobia, are well known to those skilled in the art.

[0046] NolA insensitive strains can be isolated using a variety ofselection procedures. For example, since NolA inducers inhibitnodulation, one can select for NolA insensitive B. japonicum mutants byinoculating plants with a mutated population in the presence of the NolAinducer (e.g., IND-1, IND-2, or CDF, or quorum sensing factor). Bacteriaisolated from nodules that form rapidly on the soybean roots would bepresumptive mutants that were insensitive to the inhibitory effects ofthe nolA inducers. These mutants could then be confirmed by directlytesting the ability of the inducers to activating transcription of nolA(e.g., using either Northern hybridization or measuring nolA-lacZexpression). Similarly, since nolA expression increases with cultureage, plating of mutated B. japonicum cells (containing the nolA-lacZfusion) on medium containing X-GAL(5-bromo-4-chloro-3-indolyl-β-D-galactoside) allows one to distinguishthe blue, NolA expressing, and white, NolA non-expressing, cells. Thissystem has been used to isolate and select mutants that are insensitiveto the quorum sensing inducer that is expressed in the colonies afterprolonged growth (i.e., cells remaining white).

[0047] This same selection scheme can also be used to isolate B.japonicum mutants that lack the ability to produce quorum sensingfactor. These mutants should also appear white after prolonged growth.These mutants can also be selected by plating a mutated population of B.japonicum and then overlaying these colonies with soft agar (0.4%)containing a B. japonicum strain with the nolA-lacZ fusion and X-GAL.Mutants defective in production of the quorum sensing factor will notinduce the nolA-lacZ fusion in the overlay, while those still producingthe factor will rapidly induce the fusion resulting in a blue color.

[0048] The subject invention advantageously provides methods ofincreasing nitrogen fixation in plants by applying a nodulationinoculant having NolA^(INS) mutants and one or more nolA inducers toplants. In a preferred embodiment, the plants are legumes; in a morepreferred embodiment, the plants are soybeans. The inoculant containsNolA^(INS) mutants in amounts effective to induce nodulation in theplant and amounts of one or more nolA inducers sufficient to induce theactivity of the nolA gene. Methods of preparing inoculants, or coatingseeds with inoculants, suitable for use in the present invention arewell known in the art and include those taught in U.S. Pat. Nos. 4,535,061, 5,173,424, 5,695,541, and 5,916,029 hereby incorporated byreference in their entireties.

[0049] The subject invention also provides methods of producing anodulation inoculant containing reduced amounts of quorum factor (CDF).These improved nodulation inoculants are produced by adding iron tocultures containing nodulating bacterial cells. As used herein, anodulation inoculant includes any bacterial species that nodulates aplants. Nodulation inoculants produced according to these methodscontain lower amounts of quorum factor (CDF) as compared to nodulationinoculants not grown in the presence of iron, and are able to moreefficiently nodulate target plant species (as compared to indigenousnodulating bacterial cells or nodulation inoculants not grown in thepresence of iron).

[0050] The subject invention further provides methods of reducing theproduction of cell density factor or quorum factor in a nodulationinoculant or a method of increasing the nodulation efficiency of anodulation inoculant comprising the addition of iron to mediumcontaining the nodulation inoculant. Iron is added in amounts sufficientto suppress the production of cell density factor or quorum factor.

[0051] In some embodiments of the above-identified methods, the iron isin the form of compounds containing Fe³⁺. One embodiment provides ironin the form of FeCl₃. As would be apparent to one skilled in the art,nodulation inoculants can be prepared by culturing the bacterial cellsin any size container. For example, the cells can be cultured in afermenter, batch cultured, cultured on solid medium, cultured instandard culture flasks, or cultured in test tubes.

[0052] In various embodiments, iron is added to the culture medium atvarious stages of bacterial growth in amounts sufficient to suppress theproduction of CDF or quorum factor. Thus, iron can be added tonodulation inoculants in lag, early exponential, exponential, lateexponential, early stationary, or stationary growth phase. In otherembodiments, the iron can be added to the culture medium prior to theaddition of an inoculant starter culture; alternatively, iron can beadded to the starter culture and this admixture then added to theculture medium. Iron can also be added to the culture medium and thestarter culture. Various embodiments of the invention provide for theaddition of at least about 0.05 μM or at least about 0.1 μM of iron.Other embodiments provide for the addition of iron in concentrations ofat least about 1 μM, 10 μM, 100 μM, or at least about 1 mM. Ironconcentrations that ranges from 0.5 μM to 1M can be also be used in thepractice of the instant invention. In some embodiments, the iron has aconcentration that ranges from 1 μM to 500 mM. Other embodiments provideiron concentrations that range from 10 μM to 250 mM or from 100 μM to100 mM. Alternatively, iron can be added in a range of 500 μM to 50 mM,750 μM to 5 mM, or about 1 mM. Each of these ranges is to be construedas providing written support of an iron concentration ranges fallingwithin the range. For example, the range of 100 μM to 100 mM is also tobe construed as providing written support for a ranges such as 300 μM to50 mM, 400 μM to 10 mM, or 500 μM to 1 mM. Furthermore, as would beapparent to the skilled artisan, aseptic or sterile techniques can beutilized in the practice of the invention.

[0053] In some embodiments, the nodulation inoculant comprises a singlespecies or strain of nodulating bacteria. Other embodiments provide forthe combination of different species of nodulating bacteria. Thus,combination of at least two different species of nodulating bacteria canbe used in the practice of the disclosed inventions. In someembodiments, the nodulating bacteria is one or more species or strain ofBradyrhizobium. Other non-limiting examples of inoculants that can beproduced according to the instant invention include Parasponia rhizobium(now identified as B. parasponia), Rhizobium leguminosarum biovarsviciae, trifolii and phaseoli, Rhizobium sp. NGR234, B. japonicum USDA110 and 123, Rhizobium etli, Sinorhizobium meliloti, Rhizobiumleguminosarum spp., or those listed in FIGS. 17-18.

[0054] The subject invention also provides for methods of screeningorganisms or extracts for the production of IND-1, IND-2, CDF (quorumfactor), or CDF-like molecules. In this method, extracts or culturesupernatants are analyzed for their ability to modulate nolA-lacZ,nodY-lacZ, nodC-lacZ, or nodD-lacZ fusions in transformed host cells.For example, where such molecules are present in the extract orsupernatant, nolA expression is induced. In contrast, very littleinduction is observed with samples where no IND-1, IND-2, CDF (quorumfactor), or CDF-like molecules are present. Conditioned medium fromorganisms to be tested for the presence of CDF or CDF-like molecules canalso be used in the subject screening methods.

[0055] A further aspect of the subject invention relates topolynucleotides encoding nolA inducers of the subject invention. Thepolynucleotide sequence encoding the nolA inducers may, optionally, beoperably linked to transcriptional control sequences. As is apparent toone of ordinary skill in the art, the disclosed inducers may be encodedby multiple polynucleotide sequences because of the redundancy of thegenetic code. It is well within the skill of a person trained in the artto create these alternative DNA sequences encoding the same, oressentially the same, proteins. As used herein, reference to“essentially the same” sequence refers to sequences which have aminoacid substitutions, deletions, additions, or insertions which do notmaterially affect biological activity of the inducers of the invention(namely the ability to induce nolA). Fragments of the inducers whichretain the ability to induce nolA expression are also included in thisdefinition.

[0056] The polynucleotides of the subject invention include vectors andexpression cassettes. The vectors and expression cassettes may containtranscriptional control sequences which are operably linked topolynucleotide sequences encoding the nolA inducers of the instantinvention. The vectors and expression cassettes of the invention mayfurther include selectable markers.

[0057] The subject invention also provides transformed plant cells andtransgenic plants which have one or more polynucleotide sequences whichencode plant-derived or bacterial-derived nolA inducers. Thepolynucleotide sequences encode compounds which induce the expression ofnolA, thereby reducing nodulation in plants by susceptible bacteria.Methods of transforming cells with polynucleotide sequences, vectors, orexpression cassettes which encode nolA are well known to those skilledin the art. Plants and plant cells may be transformed by, for example,electroporation, Agrobacterium transformation, engineered plant virusreplicons, electrophoresis, microinjection, micro-projectilebombardment, micro-LASER beam-induced perforation of cell wall, orsimply by incubation with or without polyethylene glycol (PEG).

[0058] The method of increasing nitrogen fixation in plants, to whichthe NolA^(INS) mutants are applied, may be practiced in transgenicplants which express the nolA inducer and non-transgenic plants whichconstitutively express the NolA inducer; this method may involve theapplication of compositions having NolA^(INS) mutants (bacterial cells)directly to the roots of transgenic plants having polynucleotidesencoding a NolA inducer. The compositions having NolA^(INS) mutants may,optionally, further include one or more NolA inducers. In oneembodiment, the roots may be wounded to enable the NolA^(INS) bacterialcells to penetrate the roots more quickly and easily; however, woundingof the roots is not required. In a preferred embodiment, the plants arelegumes. More preferably, the plants are soybeans.

[0059] The present invention also provides methods of reducing orinhibiting the nodulation activity of indigenous B. japonicum by addinga composition having one or more NolA inducers of the invention to soil.In this aspect of the invention, NolA^(INS) bacterial cells may,optionally, be included in the composition. The soil to which thesecompositions are added include active and fallow fields.

[0060] To facilitate understanding of the invention, a number of termsare defined below. All publications, patents and patent applicationscited herein, whether supra or infra, are hereby incorporated byreference in their entirety to the extent that the reference is notinconsistent with the teachings provided herein. As used in thisspecification and the appended claims, the singular forms “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise.

[0061] As used herein, the term “transgenic plants” refers to plants(monocots or dicots), having plant cells in which heterologouspolynucleotides, such as those encoding plant or bacterial nolAinducers, are expressed as the result of manipulation by the hand ofman.

[0062] As used herein, the term “peptide” refers to a polymer of aminoacids and does not refer to a specific-length of the product; thus,polypeptides, oligopeptides, and proteins are included within thedefinition of peptide. This term also does not refer to, or exclude,post expression modifications of the peptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, peptides containing one or moreanalogs of an amino acid (including, for example, unnatural amino acids,etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

[0063] The terms “purified” and “isolated” indicate that the molecule ispresent in the substantial absence of other molecules of the same type.The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofmolecules of the same type are present.

[0064] The terms “purified” and “isolated”, when referring to apolynucleotide, nucleotide, or nucleic acid, indicate a nucleic acid thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three separate genes. The term thereforecovers, for example, (a) a DNA which has the sequence of part of anaturally occurring genomic DNA molecules but is not flanked by both ofthe coding or non-coding sequences that flank that part of the moleculein the genome of the organism in which it naturally occurs (e.g., DNAexcised with a restriction enzyme); (b) a nucleic acid incorporated intoa vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in mixtures of (i) DNA molecules, (ii) transfected cells,and (iii) cell clones, e.g., as these occur in a DNA library such as acDNA or genomic DNA library.

[0065] The term “polynucleotide” as used herein refers to a polymericform of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule and thus includes double- and single-stranded DNA and RNA.It also includes known types of modifications, for example, labels whichare known in the art, methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, intemucleotidemodifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as proteins (includingfor e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmnodified formsof the polynucleotide.

[0066] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

[0067] As used herein, the term “expression cassette” refers to amolecule comprising at least one coding sequence operably linked to acontrol sequence which includes all nucleotide sequences required forthe transcription of cloned copies of the coding sequence and thetranslation of the mRNAs in an appropriate host cell. Expressioncassettes can include, but are not limited to, cloning vectors,specifically designed plasmids, viruses or virus particles. Thecassettes may further include an origin of replication for autonomousreplication in host cells, selectable markers, various restrictionsites, a potential for high copy number and strong promoters.

[0068] By “vector” is meant any genetic element, such as a plasmid,phage, transposon, cosmid, chromosome, virus etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

[0069] In order to provide a means of selecting transformed plant orbacterial cells, the vectors for transformation will typically contain aselectable marker gene. Marker genes are expressible DNA sequences whichexpress a polypeptide which resists a natural inhibition by, attenuates,or inactivates a selective substance. Examples of such substancesinclude antibiotics and, in the case of plant cells, herbicides.Suitable marker genes for use in this invention are well known to thoseskilled in the art.

[0070] It is also contemplated that a particular amino acid sequence ofNolA may be encoded by more than one polynucleotide sequence. It may beadvantageous to produce nucleotide sequences possessing a substantiallydifferent codon usage. Codons can be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence without altering theencoded amino acid sequence include the production of RNA transcriptshaving more desirable properties, such as a longer half-life, thantranscripts produced from the naturally occurring sequence.

Materials and Methods

[0071] Bacterial Stains, plasmids and culture conditions. For routinebacterial growth, B. japonicum cells were maintained on RDY medium (So,J. -S. et al. [1987] Mol. Gen. Genet. 207:15-23). Bacteria were grown inminimal medium (Bergensen, F. J. [1961] Aust. J. Biol. Sci. 14:349-360)for β-galactosidase activity. As required, antibiotics were used at thefollowing concentrations, Cm (30 μg/ml), Sm (100 μg/ml), Sp (100 μg/ml),Tc (100 μg/ml). The B. japonicum strains used in this study wereBj110-42, BJAlac12, BJAlac23, BJAlac13 and BJ110-1248-1, ZB977, SL101and Bj110-573. These strains harbored the following translationalfusions; BJ110-1248-1 (nodD₂-lacZ, plasmid pRJ1248, Dockendorff et al.[1994] Mol. Plant-Microbe Interact. 7:596-602), ZB977 (nodY-lacZ,plasmid pZB32, Banfalvi et al. [1988] Mol. Gen. Genet. 214:420-424),SL101 (npt-lacZ, Yuen, J. P. -Y and G. Stacey [1996] Mol. Plant-MicrobeInteract. 9:424-428), and Bj110-573 (nodC-lacZ, chromosomally integratedfusion, Dockendorff et al. [1994] Mol. Plant-Microbe Interact7:596-602). Strains Bj110-42, BJAlac23, BJAlac12, BJAlac13 harborednolA-lacZ translational fusions encoded on plasmids pBGALac1, pNMAlac23,pNMAlac13, pNMAlac12 respectively (Garcia et al. [1996] Mol.Plant-Microbe Interact. 9:625-635; Loh et al. [1999] J. Bacteriol.181:1544-1554). Plasmid pNMAlac23 contained mutations to ATG2 and ATG3of nolA and allowed the specific expression of NolA₁-lacZ. In contrast,plasmids pNMAlac13 (mutations to ATG1 and ATG3) and pNMAlac12 (mutationto ATG1 and ATG3, Loh et al. [1999] J. Bacteriol. 181:1544-1554) onlyexpressed NolA₂-lacZ and NolA₃-lacZ, respectively.

[0072] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted. All references, publications, and patentscited herein are hereby incorporated by reference in their entireties.

Example 1

[0073] Identification of NolA inducers from plant extracts

[0074] While many of the nodulation genes of B. japonicum are induced bythe plant flavonoids genistein and daidzein, these compounds and avariety of other flavonoids failed to induced nolA expression. NolAexpression was, however, induced by plant extracts. Analysis of theseextracts, using Reverse Phase HPLC, have identified the presence of twodistinct compounds (IND-1 and IND-2) that are capable of inducing nolA(FIG. 1).

[0075] IND-1 has been identified as a phthalic acid bis-(2-ethyl-hexyl)ester (FIG. 2A). To confirm the activity of this compound, phthalic acidbis-(2-ethyl-hexyl) ester was chemically synthesized and shown to beable of inducing nolA expression (FIG. 2B). This compound is a stronginducer of NolA expression and inhibits nodulation.

[0076] Initial analysis of IND-2 indicates that its activity issensitive to chitinase treatment. IND-2 may also be purified fromsoybean extracts, particularly commercially available soybeanphosphatidylinositol extracts (available from companies such as SigmaChemical Co., St. Louis, Mo.). When tested, the commercially availablephosphatidylinositol soybean extracts (Sigma) were found to be capableof inducing nola expression (FIG. 3). Moreover, these extracts were alsosensitive to chitinase treatment. As shown in FIGS. 4A-4B, we haveidentified a peak (i.e. peak 9) after Reverse Phase HPLC, that is bothcapable of inducing nola and sensitive to chitinase. Materialscontaining IND-1 and IND-2 were applied to a C18 column (Phenomenex,Inc., Torrance, Calif.) and eluted with a methanol gradient (0-100%) ata flow rate of 1 mL per minute.

Example 2

[0077] NolA inducer from B. japonicum

[0078] Typically, B. japonicum cells are found in high populationdensity in commercial inoculants. To determine if the bacterial nolAinducer was present in commercial inoculant, three soybean inoculantsfrom two commercially available sources were extracted with butanol, andthese extracts analyzed for their ability to induce the nolA-lacZfusions. As shown in Table 1, nolA expression was induced significantlyby the B. japonicum inoculant extracts. In contrast, very littleinduction was observed with samples where no B. japonicum were present(i.e., peat alone).

Example 3

[0079] Quorum control mechanism for NolA induction

[0080] NolA expression is population density dependent; its expressionis low at a low population density, and significantly higher in moredense cultures (FIG. 5). This quorum control of NolA expression appearsto be regulated by a compound that is secreted and accumulates in theculture medium. Addition of this compound (i.e. conditioned medium) toB. japonicum cultures grown to a low population density greatlyincreases the expression of nolA (Table 2). Consistent with the factthat nolA regulates nodD₂, the levels of nodD₂ expression not onlyshowed a similar population density dependence (FIG. 6, Table 2), butwere also found to be affected by the addition of the NolA inducer.

[0081] As shown in Tables 2-3 and FIG. 11C, an inducer of nolA-lacZ andnodD₂-lacZ expression was detected in conditioned medium from B.japonicum cultures grown to a high population density. This inducer wascapable of inducing transcription of both fusions when added to B.japonicum cultures at 10 μl/ml. The ability of the conditioned medium toinduce the nolA fusions was population density dependent with little orno induction of the fusions observed using conditioned medium derivedfrom cultures of A₆₀₀<0.2 (FIG. 11C). Significant induction was observedwith conditioned medium from cultures of A₆₀₀=0.5, reaching a maximum atA₆₀₀>1.0. The bacterially derived inducer within the conditioned mediumis insensitive to heat. The nolA inducer present in the conditionedmedium was lost after dialysis using a membrane with a cutoff at 3 kDa(Fisher Scientific, Pittsburgh, Pa).

Example 4

[0082] Population density dependence of Nod gene expression

[0083] Nod gene induction by genistein is population-density dependent.It has been observed that optimal gene expression occurrs at very lowpopulation densities (i.e., A₆₀₀<0.05, Yuen, J. P. -Y. and G. Stacey[1996] Mol. Plant-Microbe Interact. 9:424-428). In order to examine thisobservation in a systematic way, Bj110-573 cells (containing achromosomally integrated nodC-lacZ fusion, Dockendorff, T. C. et al.[1994] MoL Plant-Microbe Interact. 7:596-602) were cultured to variouspopulation densities. Aliquots were taken from these cultures, adjustedto specific, initial population densities, and tested for nodC-lacZinduction using a suboptimal level of genistein (0.025 μM). In each ofthese experiments, cells were cultured in the presence of the inducerfor 5 hours; a time period that previous experiments had indicatedresulted in optimal nodC-lacZ expression. This experimental designallowed for the analysis of nodC-lacZ expression as a function both ofthe inoculum population density and the initial population density ofthe inducer-treated aliquots. As shown in FIG. 6, the inducibility ofnod gene expression was highest in samples where the inoculum cultureswere grown to low optical population densities. For example, an inoculumculture grown to an A₆₀₀=0.068 was significantly more inducible than aculture grown to an A₆₀₀=0.852 regardless of the density at which thesecells were assayed. Little or no nodC induction was observed with cellsharvested at high population densities (e.g., A₆₀₀=1.62 or 3.22).

[0084] A similar dependence on population density was also observed whenthe levels of nod gene expression were examined relative to the densityof the cell suspension treated with inducer. For example, cellsharvested at an A₆₀₀=0.192 showed very high levels of nodC-lacZexpression when induced at an A₆₀₀=0.1 but inducibility was markedlyreduced when the same cells were assayed at an A₆₀₀=0.3. These resultssupport the notion that the responsiveness of B. japonicum cells to thenod gene inducer, genistein, is affected by culture age, but is moredirectly related to culture population density.

Example 5

[0085] Nod gene expression

[0086] nodY-lacZ induction by genistein was highest at a low populationdensity and drastically reduced at high cell cultures. The fact thatnola is involved in the negative regulation of the nod genes and thatits expression increased in cultures of higher population density,suggested a role for this protein in population density dependent nodgene repression. This view is supported experimentally by the followingtwo results. First, nodY-lacZ expression in a nola mutant was found tobe unaffected by population density (FIG. 7). Moreover, when thepopulation density dependent factor was added to B. japonicum culturesgrown to a low population density, the levels of nod gene induction byisoflavonoids were significantly reduced (Table 2). It is, therefore,likely that high population densities coupled with increased nolaexpression in response to a bacterial quorum factor leads to elevatedexpression of the repressor NodD₂; this results in negative control ofthe flavonoid inducible nod genes.

Example 6

[0087] Effect of IND-1 on nod gene expression

[0088] The levels of nod gene expression were also found to be affectedby IND-1. Similar to the bacterial factor, incubation of IND-1 with B.japonicum cells reduced nodY-lacZ induction by genistein (FIG. 8). Thisinhibition was observed in three B. japonicum strains tested (i.e.,USDA110, USDA76, USDA31), suggesting a general effect of nod generepression by IND-1. When further analyzed, we noted that both thequorum factor and IND-1 appeared to affect only NolA, expression, butnot NolA₂ or NolA₃ (Table 2, FIG. 9).

Example 7

[0089] Effect of IND-1 and quorum factor on nodulation

[0090] The significance of nod gene repression by both IND-1 and thequorum factor was also investigated in plant tests. Seeds of Glycine maxcv Essex were surface sterilized, placed in sterile growth pouches (MegaInternational, MN) and cultivated as described by Nieuwkoop et al.(Nieuwkoop et al. [1987] J. Bacteriol. 169:2631-2638). Each seedling wasinoculated with 10⁷ B. japonicum cells. Prior to inoculation of theroots, B. japonicum cells were incubated for 1 h with concentrated,conditioned medium that had been sterilized by filtration through a 0.45μM filter (Millipore, Bedford, Mass.). The conditioned medium was usedat a final concentration of 10 μl per 1 ml of culture. At the time ofinoculation, the location of the root tip was marked on the outside ofthe plastic growth pouch. Plants were watered with nitrogen freenutrient solution (Wacek and Brill [1976] Crop Sci. 15:519-523). At 21days post-inoculation, the number of nodules on each root both above andbelow the root tip mark was recorded.

[0091] Consistent with the fact that the nodulation genes are criticalfor effective nodulation, pretreatment of B. japonicum cells with eitherIND-1 or the quorum factor resulted in reduced nodulation efficiency.This is shown in FIG. 10, where a delay in nodulation in both IND-1 andquorum factor treated samples, is reflected by an increased number ofnodules on both lateral roots as well as below the RT mark (i.e. roottip mark at the time of inoculation).

[0092] Such a delay in nodulation is significant in light of resultspresented above that demonstrate that the quorum determinant is alsopresent in commercial preparations used as soybean inoculants. In theseinoculants, the levels of quorum factor would be present in sufficientlevels to repress any stimulation of the nod genes by plant producedisoflavonoids, thereby reducing the ability of B. japonicum in theseinoculants to nodulate the soybean plant.

Example 8

[0093] Cell population density dependence of NolA expression

[0094] The expression of nodD₂-lacZ and nolA-lacZ fusions as a functionof B. japonicum culture density was examined (FIG. 11A). Both nolA-lacZand nodD₂-lacZ expression exhibited a basal level of transcription untilmid-log phase (A₆₀₀=0.5) at which time expression increased to a maximumat a population density of A₆₀₀>1.0. The level of nodD₂-lacZ expressionin the NolA mutant strain BjB3 (Garcia et al. [1996] Mol. Plant-MicrobeInteract. 9:625-635) did not increase with population density andremained at background levels throughout the experiment (data notshown). Thus, these data indicate that the level of NolA expressionincreases with population density resulting in an elevated level ofNodD₂ production. As a control, the level of neomycin phosphotransferase(npt-lacZ) production was not affected by population density indicatingthat the regulation of gene expression was specific for NolA and NodD₂.

[0095] NolA₁-LacZ, NolA₂-LacZ or NolA -₃LacZ fusions were assayed as afunction of population density. The results indicated that only NolA₁expression is cell-density dependent and required for NodD₂ expression(FIG. 11B).

Example 9

[0096] Isolation of Cell Density Factor (quorum factor) from B.japonicum conditioned medium

[0097]B. japonicum conditioned medium was concentrated approximately10-fold. The material was then applied to a C18 column (Phenomenex,Inc., Torrance, Calif.) and eluted with a methanol gradient (0-100%) ata flow rate of 1 mL per minute. The HPLC elution profile is shown inFIG. 12. Cell density factor was demonstrated to be a potent inducer ofnolA expression.

Example 10

[0098] Effect of Fe³⁺ on nodulation activity

[0099] Analyses of CDF production revealed decreased levels of CDF incultures grown in the presence of iron. As shown in FIG. 14A, theaddition of Fe³⁺ to B. japonicum cultures significantly reduced theexpression of nolA expression at high culture densities. Consistent withthe fact that nolA is involved in the repression of the nodulationgenes, a corresponding increase in nodY-lacZ expression was also noted(FIG. 14B) when Fe³⁺ was added to B. japonicum cultures. These resultsindicate that Fe³⁺ can inhibit CDF production and, therefore, reduce thenegative effect of quorum regulation on B. japonicum nod geneexpression.

Example 11

[0100] Recombinantly or UV produced mutants

[0101] We have generated, by recombinant means, mutations in the nwsBgene. The NwsB mutant appears to be defective in the recognition of thequorum signal and we have performed plant tests with the mutant incompetition assays with the wild-type bacteria. In these assays, theeffect of IND-1 (BEHP) on the ability of the NwsB mutant to nodulatesoybean in the presence of wild-type bacteria (at different cell ratios)was examined. The NwsB strain should have a competitive advantage due tothe lack of nod gene repression since it does not respond to the quorumsignal. Nodule occupancy was scored both above (treated area) and below(new root growth) the root tip mark at the time of inoculation. As shownin FIG. 15, the addition of BEHP did increase the percentage of nodulesthat were occupied by the NwsB mutant. These studies were conductedusing 25 μM levels of BEHP.

[0102] Another means to generate a mutant similar to NwsB is by UVmutagenesis. A commercial strain, such as B. japonicum Bj61A273, istransformed with a conjugative plasmid containing the nolA gene fused tothe lacZ gene, encoding γ-galactosidase. The latter is a reporter enzymethat allows the detection of NolA expression by formation of blue coloron plates containing the chemical X-Gal. UV treated B. japonicum cellsharboring a nolA-lacZ fusion are screened and selected for colonies thatremain white on agar containing X-gal and inducing levels of the BEHP.To test whether an NwsB mutant has been isolated, complementationstudies with the mutant and the nwsB gene will be conducted.

[0103] Alternatively B. japonicum mutants can be isolated using thesoybean plant as the means of selecting strains that are resistant toBEHP (FIG. 16). UV treated USDA61A2 73 cells were inoculated ontosoybean roots in the presence of BEHP. Most of the nodules formed in theUV treated samples demonstrated a delayed nodule phenotype in thepresence of BEHP (ie., below the root tip mark at the time ofinoculation). Nodules formed above the root tip mark were extracted forbacteria. Single colonies have been isolated and are being re-screenedfor their ability to nodulate soybean plants in the presence of BEHP. Inaddition, these colonies are being transformed with the nolA-lacZplasmid, and will be tested for BEHP mediated nolA-lacZ activity.

Example 12

[0104] CDF-like molecules found in other organisms

[0105] Other rhizobia have been tested for the production a compoundsimilar to the B. japonicum CDF. The B. japonicum nolA-lacZ fusion wasused as a reporter system to test culture supernatants from a variety ofbacteria. The assay system has been described above in Examples 2-6.

[0106] As shown in FIG. 17, we found that supernatants from rhizobialstrains tested were able to induce the nolA-lacZ fusion. This indicatesthat compounds similar to CDF present in these cultures. R. lupini andS. meliloti were assayed for, and demonstrated to possess, a CDF-likemolecule by examining the HPLC retention times of the active component(data not shown) in culture supernatants. In both cases, the activecomponent exhibited a similar retention time to the B. japonicum CDF.

[0107] The presence of CDF-like molecules expressed by other bacteriahas been analyzed using nolA-lacZ expression systems as a bioassay.These results are shown in FIG. 18.

[0108] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application. All documents, patents, patent applications, andreferences cited within this application are hereby incorporated byreference in their entireties. TABLE 1 Induction of nolA-Lac expressionby extracts of commercial inoculants. β-galactosidase activity (U)Untreated Urbana Urbana Uninduced peat Labs #J168 Labs #196 Lipha Tech83 ± 22 109 ± 18 438 ± 10 521 ± 43 356 ± 26

[0109] TABLE 2 Expression of nolA- and nodD₂-lacZ fusions in response tothe population density dependent factor β-galactosidase activity (U)Uninduced CM^(a) CM^(b) (heat) LacZ fusion NolA_(1,2,3)-lacZ 22 ± 2 300± 25 292 ± 22 NolA₁-LacZ 35 ± 3 205 ± 19 225 ± 18 NolA₂-lacZ 24 ± 4 42 ±5 39 ± 5 NolA₃-lacZ 38 ± 6 62 ± 4  59 ± 308 NodD₂-lacZ  58 ± 14 393 ± 11356 ± 16

[0110] TABLE 3 Effect of population density dependent factor on theinduction of nodY-lacZ in B. japonicum by genistein β-galactosidaseactivity (U) genistein^(c) genistein^(c) Uninduced genistein^(c) +CM^(a) + CM^(ab (heat)) 3 ± 0 366 ± 11 33 ± 5 23 ± 5

We claim:
 1. A method of producing a nodulation inoculant containingreduced amounts of cell density factor (CDF) comprising the addition ofiron to growth medium for a nodulation inoculant in amounts sufficientto reduce the concentration of CDF.
 2. A method of screening an extractor cell culture supernatant for the presence of an IND-1, IND-2, CDF, orCDF-like compound comprising: a) obtaining an extract or cell culturesupernatant; b) contacting a host cell transformed with one or moregenetic constructs containing a reporter enzyme selected from the groupconsisting of nolA-lacZ, nodY-lacZ, nodC-lacZ, or nodD-lacZ with saidextract or cell culture supernatant; and c) analyzing the contacted hostcell for the modulation or expression of said nolA-lacZ, nodY-lacZ,nodC-lacZ, or nodD-lacZ reporter enzyme.
 3. The method according toclaim 1, wherein said iron is Fe³⁺.
 4. The method according to claim 1,wherein said nodulation inoculant comprises Bradyrhizobium species. 5.The method according to claim 1, wherein said nodulation inoculantcomprises Bradyrhizobium japonicum.
 6. The method according to claim 1,wherein medium is liquid.
 7. The method according to claim 1, whereinsaid iron is added prior to the addition of the nodulation inoculant. 8.The method according to claim 1, wherein said iron is addedsimultaneously with the nodulation inoculant.
 9. The method according toclaim 1, wherein said iron is added after the nodulation inoculant. 10.The method according to claim 1, wherein said iron is added to thenodulation inoculant and the iron containing inoculant is added to themedium.
 11. The method according to claim 1, wherein said iron isseparately added to the nodulation inoculant and the medium.
 12. Themethod according to claim 1, wherein the iron has a concentration of atleast about 0.5 μM or at least about 0.1M.
 13. The method according toclaim 1, wherein the iron has a concentration that ranges from 0.5 μM to1M.
 14. An isolated compound selected from the group consisting of IND-2and CDF.
 15. A composition comprising a soil additive or conditioner anda compound selected from the group consisting of IND-1, IND-2, and CDF16. The composition according to claim 22, wherein the compound is IND-1(bis-ethyl-hexyl-ester phthalate).
 17. An isolated bacterial celldefective in recognition of NolA inducer compounds.
 18. The isolatedbacterial cell according to claim 21, wherein said bacterial cellcontains a defect in the nwsB gene.
 19. A method of suppressing thenodulation activity of indigenous nodulating bacterial cells comprisingthe addition of one or more NolA inducers to soil containing saidindigenous nodulating bacterial cells.
 20. The method according to claim19, wherein said NolA inducer is bis-ethyl-hexyl-ester.
 21. Acomposition comprising a carrier and a nodulation inoculant producedaccording to the process of claim 1.